1. Field of the Invention
The invention in the field of biochemistry and neurosciences relates to compositions and methods based on the neuregulin heparin binding domain (N-HBD) use to target other polypeptides to cell surfaces and extracellular matrix rich in heparan sulfate proteoglycans (HSPGs) for the treatment of cancer or various nervous system diseases.
2. Description of the Background Art
In order to carry out their diverse physiological functions, cells must adhere to cellular and extracellular components of their environment in a specific manner. Adherence-requiring functions in the nervous system include neurite extension, synapse formation and axon myelination. The ability to recognize multiple environmental cues and undergo specific adhesive reactions is critical to such complex cellular functions. Recognition and adhesion are mediated by cell adhesion molecules (“CAMs”) which bind to macromolecules expressed on neighboring cells or in the extracellular matrix (“ECM”).
Three motifs present in adhesion molecules for which 3D structure is known are: immunoglobulin (Ig) superfamily domains, fibronectin type III (Fn-III) domains and the domains found in cadherins. In the nervous system, Ig superfamily members mediate Ca-independent homophilic and heterophilic binding. The extracellular regions of these molecules include one or more domains with sequence similarity to variable (V) or constant (C) domains of antibodies, i.e., Ig's (Williams, A F et al., Annu. Rev. Immunol. 6:381-405 (1988). Yoshihara, Y et al., Neurosci. Res. 10:83-105 (1991). Many Ig superfamily molecules consist of tandem Ig-like domains bonded in series with multiple copies of a second building block domain (e.g., an Fn-III repeat). Because two molecules that share detectable sequence similarity have been found to adopt the same folding topology, investigators have used structures of molecules discovered in studies of the immune system as “first order” models for the structures of Ig domains in neural CAMs.
These molecules, in particular Ig-like domains, and their topology are reviewed in detail in Vaughn D E et al., Neuron 16:261-273 (1998), which is incorporated by reference in its entirety. Ig V domains are the prototype of the V-like domains of CAMs and their type of fold is found in antibody VH and VL domains and the N-terminal domains of the T cell receptor α Vaughn et al., supra, V-like domains are found in T cell surface molecules CD4 (first and third domains) and CD8, the N-terminal domains of the “immune system CAM” CD2, vascular cell adhesion molecule-1 (VCAM-1) and telokin, and the C-terminal domain of the myosin light chain kinase. Ig C1 domains consist of seven B strands arranged into two antiparallel sheets. The two sheets are connected by a disulfide bond between strands “B” and “F.” In an antibody, constant domains are found in the Fc regions and the C-terminal domains of the Fab Ig fragment. Constant-like or C1 set domains, are also found in the membrane proximal domains of MHC molecules and T cell receptors. The C2 and C1 folding topologies are similar, except for the “sheet switching” of one β strand and the fact that β strands in Ig C2 domains are somewhat shorter (˜6 vs ˜9 residues) and lack many of the conserved sequence patterns at the N-terminal end of the β barrel. According to Vaughn et al., supra, C2 domains are present in three Ig superfamily members: CD2, the second domain of VCAM-1 and the second and fourth domains of CD4. The heparin binding domain of neuregulin, which is at the center of the present invention, is an Ig-C2 domain.
An important means of intercellular communication is the release of growth and differentiation factors from one cell and their binding to and activation of membrane receptors on a nearby cell which ultimately changes its properties through changes in gene expression. Once released, many polypeptide factors have additional binding interactions with heparan-sulfate proteoglycans (HSPGs) situated in the ECM between cells. The functional consequences of this dual-binding interaction are not entirely clear, but may serve to concentrate these factors at sites where they are needed, to protect them from proteolysis, and to modulate their interactions with their receptors (Schlessinger, J. et al., (1995) Cell 83, 357-360). What is even less clear, is how these extracellular interactions modulate the intracellular events that ultimately change a cell's properties.
The neuregulins (NRGs) are a family of heparin-binding growth and differentiation factors with multiple functions in (a) growth and development of the nervous system and heart, and (2) cancer (Fischbach, G. D. et al. (1997) Annu Rev Neurosci 20, 429-458). NRGs are released from motor nerve endings at neuromuscular synapses and activate members of the epidermal growth factor (EGF) family of tyrosine kinase receptors, erbB2, erbB3 and erbB4, in the postsynaptic muscle membrane (Loeb, J A et al., (1999) Development 126, 781-791; Goodearl, A D et al., (1995) J Cell Biol 130, 1423-1434; Moscoso, L M et al., (1995) Developmental Biology 172, 158-169; Zhu, X et al., (1995) EMBO Journal 14, 5842-5848). This trans-synaptic activation results in a dramatic up-regulation of muscle acetylcholine receptors (AChRs) needed to guarantee proper synaptic transmission. NRG also promotes the transition from embryonic to adult forms of mammalian AChRs by inducing the switch to the α-AChR subunit (Martinou, J C et al., (1991) Proc. Natl Acad Sci USA 88, 7669-7673) and the expression of voltage-gated sodium channels (Corfas, G et al., (1993) Journal of Neuroscience 13, 2118-2125). All of these effects are expected to increase the efficacy of synaptic transmission as the target muscle fiber increases in size and the neuromuscular junction matures. Consistently, mice in which the Type I NRG1 allele is disrupted by homologous recombination, exhibit (1) a 50% reduction in the density of postsynaptic AChRs, and (2) a reduced safety factor for neuromuscular transmission when challenged by low doses of curare (Sandrock, A W et al. (1997) Science 276, 599-603).
A common feature shared by all NRGs is an epidermal growth factor-like (EGF-like) domain. Even when expressed by itself, this domain is sufficient for receptor binding and activation of homo- and heterodimers of erbB2, erbB3, and erbB4 receptors which are highly concentrated at the neuromuscular synapses in the postsynaptic muscle membrane (Moscoso et al.,supra; Xu et al., supra; Altiok, N. et al., v (1995) Embo J 14, 4258-4266). The rapid autophosphorylation of Tyr residues in these receptors initiates a signaling cascade that translates the initial binding event into the induction of AChR genes (Corfas, G. et al., (1993) Proc. Natl Acad Sci USA 90, 1624-1628). This signaling cascade involves a number of signaling pathways including both the mitogen-activated protein (MAP) kinase (Si, J. et al., Luo, Z., and Mei, L. (1996) J Biol Chem 271, 19752-19759; Tansey, M G et al., (1996) J. Cell Biol 134, 465-476; Altiok, N et al., (1997) EMBO Journal 16, 717-725) and phosphatidyl-inositol 3-kinase (PI3K) pathways (Si et al., supra).
Most spliced forms of NRG also have an immunoglobulin-like (IG-like) domain N-terminal to the EGF-like domain (FIG. 1). Because this domain is a heparin-binding domain (“HBD”) it is referred to herein as the neuregulin HBD (or “N-HBD”). The terms “IG-like domain” (from NRG) and “N-HBD” are meant to be interchangeable.
The present inventor and others have shown that this domain interacts with HSPGs and may lead to the deposition of NRGs in the ECM of neuromuscular synapses and within the central nervous system (Loeb et al., supra; Loeb, J A et al., (1995) J Cell Biol 130, 127-135; Meier, T., et al., (1998) J Cell Biol 141, 715-726). HSPGs, including agrin, have been identified to play important roles in neuromuscular junction formation (Sanes, J R et al., (1999) Annu Rev Neurosci 22, 389-442).
HSPGs may serve to “direct” the accumulation of NRG forms that include the N-HBD to the basal lamina of developing neuromuscular synapses and to other locations in the developing nervous system at key stages of development (Loeb et al., 1999, supra). The functional consequences of NRG-HSPG interactions on AChR expression, however, are not known.
One feature of NRG that distinguishes it from other heparin-binding ligands is that it has distinct domains for heparan sulfate binding and receptor binding that are separated from one another by a glycosylated spacer region. Recognition of this fact led the present inventor to determine the direct effects of HSPG binding on receptor- and gene activation that would not be readily possible with other heparin-binding ligands.
Rio, C et al., Neuron 19:39-50 (1997) described a 27 amino acid peptide of chick NRG that corresponded to the HBD. This peptide was made only for use as an immunogen for producing an antiserum in rabbits.
Loeb, J A et al., 1995, supra, speculated that immobilization of NRGs to the ECM might be via their Ig-like domains binding to HSPGs. This was derived indirectly from the observation that heparin inhibited post-binding receptor tyrosine phosphorylation caused by recombinant NRGs.
Since NRGs bind to heparin (Falls, D L et al., 1993, Cell 72:801-815), Meier T et al., J Cell Biol, 1998, 141:715-726, examined whether recombinant HRG (=NRG) cloned from a human cDNA library bound directly to recombinant chick agrin (a HSPG) by the negatively charged glycosaminoglycan (GAG) side chains as proposed by Loeb et al., supra. It was found that the Ig-like domain of NRGs mediated binding to these GAG chains. To test whether interaction of NRGs with components of the synaptic ECM could be mediated by the Ig-like domain, the investigators expressed a truncated HRG protein containing the Ig-like domain, HRGΔBbsI and discovered that the Ig-like domain, but not the EGF-like domain, bound to agrin.
While there have been numerous disclosures of Ig-C regions or various parts of Ig molecules fused to other proteins for various purposes, these primarily derived from true Ig molecules. The N-HBD of the present invention has less than 40% homology or sequence similarity to these true Ig domains so as to be distinct structurally and functionally from those in the prior art. Examples of such disclosures include the following.
U.S. Pat. Nos. 5,116,964 and 5,428,130, (Capon, et al) disclose a nucleic acid encoding a polypeptide fusion comprising a ligand binding partner protein containing more than one polypeptide chain one of which may be fused to an Ig C region through C-terminal carboxyl or the N-terminal amino groups. The lectin domain described in these documents, which is completely distinct from the NRG-HBD neuregulin IG domain of the present invention, is said to target active peptides to cell surfaces. Moreover, such targeting is not directed to, nor specific for, heparan sulfates at the cell surfaces. U.S. Pat. No. 5,565,335 (Capon, et al.) describes an “immunoadheson” comprising a fusion protein in which a polypeptide making up the adheson variable (V) region is fused at its C-terminus to the N-terminus of a polypeptide comprising an Ig C region.
U.S. Pat. Nos. 6,018,026 and 5,155,027 (Sledziewski et al.) describe biologically active polypeptides (and their coding DNA), and, specifically, dimerized fusion products comprising a first and a second polypeptide chain, each of which comprises a non-Ig polypeptide and requires dimerization for biological activity, joined to a dimerizing protein of heterologous origin relative to the non-Ig polypeptide. Also described is a polypeptide chain of the non-Ig polypeptide dimer, joined to at least one Ig H chain C region domain (CH1, CH2, CH3 or CH4. The expressed, dimerized fusion polypeptide exhibits biological activity characteristic of the non-Ig polypeptide dimer.
U.S. Pat. No. 5,541,087 (Lo, et al.) describes DNA encoding a fusion protein comprising a polynucleotide encoding an Ig Fc region which lacks at least the CH1 domain and a target protein sequence. U.S. Pat. No. 5,869,046 (Presta, et al.) discloses a method for preparing a variant “polypeptide of interest” which is an Fab or a (Fab′)2 fragment, the Ig domain (or an Ig-like domain) of which comprises at least one of a CH1 or CL, region. U.S. Pat. No. 6,121,022 (Presta, et al.) discloses a modified polypeptide having an Ig C domain or an Ig-like C domain and an epitope that binds to a salvage receptor within the Ig- or Ig-like C domain. This epitope, absent from the unmodified polypeptide, is taken from two loops of the CH2 domain of an Ig Fc region. The Ig-like domains described in these documents are clearly distinct from the N-HBD of the present invention.
U.S. Pat. No. 6,121,415 describes a family of polypeptides, collectively called neuregulins (NRG1) that appear to result from alternate splicing of a single gene which was mapped to the short arm of human chromosome 8 by Orr-Urtreger et al (1993) Proc. Natl. Acad. Sci. USA 90:1867-1871. The NRG3s (murine and human) were disclosed as being about 713 and 720 amino acids in length, respectively, and to comprise an EGF-like domain, an N-terminal hydrophobic segment, the serine/threonine-rich portion, a predicted transmembrane domain, and a predicted intracellular domain.
Three documents by Holmes et al. (Science 256:1205-1210 (1992); WO 92/20798; and U.S. Pat. No. 5,367,060) describe isolation and cloning of a family of polypeptide activators for the HER2 receptor which they called heregulin-α (HRG-α), heregulin-β1 (HRG-β1), heregulin-β2 (HRG-β2), heregulin-β2-like (HRG-β2-like), and heregulin-β3 (HRG-β3). These documents describe (1) the ability of the purified HRG (=NRG) polypeptides to activate tyrosine phosphorylation of the HER2 receptor in MCF7 breast tumor cells and (2) the mitogenic activity of the HRG polypeptides on tumor cells expressing high levels of the HER2 receptor. Like other EGF family growth factors, soluble HRG polypeptides appear to be derived from a membrane bound precursor (pro-HRG) which is proteolytically processed to release the 45 kDa soluble form. Although substantially identical in the first 213 amino acid residues, the HRGs are classified into two major types, α and β, based on two variant EGF-like domains which differ in their C-terminal regions. Based on an amino acid sequence comparison, Holmes et al., supra found that between the first and sixth cysteines in the EGF-like domain, HRGs were 45% similar to heparin-binding EGF-like growth factor (HB-EGF), 35% identical to amphiregulin, 32% identical to TGF-α, and 27% identical to EGF.
Falls et al. (1993) Cell 72:801-815 described another heregulin family member which termed “acetylcholine receptor inducing activity” (ARIA) polypeptide. The chicken-derived polypeptide stimulated synthesis of muscle AChR s. See also WO 94/08007. ARIA is a β type HRG and lacks the entire spacer region rich in glycosylation sites between the Ig-like domain and EGF-like domain of HRGα, and HRGβ1-β3.
Marchionni et al. (1993) Nature 362:312-318, identified several bovine-derived proteins named glial growth factors (GGFs) which share the Ig-like domain and EGF-like domain with the other NRG/HRG proteins described above, but also have an amino-terminal kringle domain. See also WO 92/18627; WO 94/00140; WO 94/04560; WO 94/26298; and WO 95/32724.
Ho et al.(1995) J. Biol. Chem. 270:14523-14532, described another member of the HRG family called sensory and motor neuron-derived factor (SMDF) which has an EGF-like domain characteristic of all other HRG polypeptides but a distinct N-terminal domain. The major structural difference between SMDF and the other HRG polypeptides is the lack of an Ig-like domain and the “glyco” spacer characteristic of all the other HRG polypeptides.
Caraway et al. (1994) J Biol Chem. 269):14303-14306 subsequently demonstrated that ErbB3 is a receptor for HRG and mediates phosphorylation of intrinsic tyrosine residues as well as phosphorylation of ErbB2 receptor in cells which express both receptors. HRG was the only known member of the EGF-like family that could interact with several receptors (Carraway et al. (1994) Cell 78:5-8.
A number of biological activities of the NRG/HRG proteins have been described:                (1) myotube differentiation by acting on synthesis and concentration of neurotransmitter receptors in the postsynaptic muscle (Falls et al., supra);        (2) increased number of sodium channels in chick muscle (Corfas et al., (1993) J. Neuroscience 13:2118-2125);        (3) mitogenic stimulation of subconfluent quiescent human myoblasts and their differentiation to yield more myotubes (Sklar et al. (1994) J. Cell Biochem. Abstr. W462, 18D, 540); and WO 94/26298, Nov. 24, 1994); and        (4) NRG1, expressed in endocardium, is an important ligand required for activation of myocardial ErbB2 and ErbB4 receptors (Ford, B D et al., Dev Biol. (1999) 214:139-150; Carraway, K L et al., Bioessays (1996) 18:263-266.        